Since oHSV's replicative ability is one of the variables that contribute to its antitumor efficacy, we have been searching for pharmacologic modulators that would enhance it. Our published data has found that histone deacetylase inhibitors (HDACi), such as valproic acid (VPA), enhance replication of oHSV in glioma cell lines. In our pilot data, we have found that this is valid, even in freshly excised gliomas, cultured as spheroids (GSs) in serum-free media in the presence of defined growth factors. In order to discover the reason for this, we have employed a specific inhibitor of one ofthe HDACs (HDAC6) and in pilot experiments have shown that we can replicate the effect of VPA, known to be a pan-HDACi. Interestingly, both specific inhibition of HDAC6 and pan-inhibition of HDACs revert type 1 interferon's (IFN) antiviral effect. Our pilot data appear to show a novel mechanism of antiviral signaling that routes incoming oHSVs to autophagosomes for xenophagy rather than to nuclei for replication. To confirm the role of HDAC6 as a novel antiviral signal and its inhibition of IFN antiviral action, we thus propose to: 1- Determine if inhibition of HDACS leads to enhanced oHSV replication in a panel of freshly excised human glioma spheroids (GSs) in vitro and in vivo, 2- Determine whether there is cross-talk between type 1 interferon signaling and HDAG6 activity in oHSV infected glioma cells and GS, and 3- Determine whether the observed antiviral function of HDAC6 relates to its action on oHSV endocytic trafficking. To perform this project, Core C will provide us GSs, Core B will provide purified lots of oHSVs, and Core A will provide the necessary biostatistical support. We will need to interact with Project 4 (Caligiuri) who has also discovered that HDACi inhibits NK cell generation of IFNy to correlate with our observed HDAC6 inhibition leading to improved oHSV replication and with Project 1 (Glorioso) and Dr. Cripe (collaborator) to determine the role of oHSV receptors and glioma receptor targeting in improving-entry and HDAC6-dependent post-entry events. We also provide critical information to Project 1 (Glorioso) for improvements in oHSV design and to Project 3 (Kaur) to determine whether HDACi will enhance replication of Chase-expressing oHSVs.